MICROTUBULE/ACTIN INTERACTIONS IN CELL MOTILITY

细胞运动是发育、伤口愈合和组织重塑的关键，也是癌症转移过程中的核心问题。因此，在基本水平上了解细胞运动是很重要的。细胞骨架聚合物、微管和丝状肌动蛋白（f-actin）是细胞运动所必需的。极化的f-肌动蛋白组装和运动被认为驱动细胞运动，而微管被认为在空间上指导f-肌动蛋白组装的位置。有证据表明，微管可能通过Rho家族的小gtp酶激活细胞信号来实现这一目标。然而，微管的组织和动力学以及它们如何受到肌动蛋白的影响尚未完全表征，并且在细胞运动过程中参与介导它们相互作用的分子是未知的。我从3个层面来解决这些问题：首先，通过使用多模式显微镜检查活细胞中的微管和f-肌动蛋白动力学，通过显微镜和生物化学探索细胞提取物模型系统中的微管/f-肌动蛋白相互作用，最后通过检查体外运动机制纯化组分的生物化学相互作用。独特的显微镜方法和分子生物化学的结合是一种新的方法，可以仔细地表征细胞现象，然后直接到试管中检验关于现象背后的分子机制的假设。在这个提议中，我描述了三个具体的目标，以阐明我们对细胞运动中微管/肌动蛋白相互作用的理解。1. 开发新的显微镜方法，用于同时可视化和分析f-肌动蛋白和微管动力学，并仔细分析活迁移细胞中微管/肌动蛋白的相互作用。2. 目的：探讨爪蟾卵提取物中微管/肌动蛋白相互作用的分子机制。3. 生化分析微管介导Rho家族信号，研究生长微管在细胞运动过程中激活Rac1的机制。这些研究结果将大大提高我们对细胞运动的机制和力学的理解，并将为分析活细胞中的蛋白质动力学提供新的方法。

Cell locomotion is critical to development, wound healing and tissue remodeling, and a central problem during metastasis in cancer. Thus, it is important to gain an understanding of cell motility at the basic level. The cytoskeletal polymers, microtubules and filamentous actin (f-actin) are required for cell motility. Polarized f-actin assembly and movement is thought to drive cell motility, while microtubules are believed to spatially direct sites of f-actin assembly. There is evidence that microtubules may achieve this by activation of cellular signaling via small GTPases of the Rho family. However, the organization and dynamics of microtubules and how they are affected by actin is not thoroughly characterized, and the molecules involved in mediating their interactions during cell motility are unknown. I approach these problems at 3 levels: First, by examining microtubules and f-actin dynamics in living cells using multimode microscopy, by exploring with both microscopy and biochemistry microtubule/f-actin interactions in a cellular extract model systems, and finally by examining the biochemical interactions of purified components of the motile machinery in vitro. The combination of unique microscopy methods and molecular biochemistry is a novel means of carefully characterizing cellular phenomenon and then going directly to the test tube test hypotheses about the molecular mechanisms underlying the phenomena. In this proposal, I describe three specific aims to shed light on our understanding of microtubule/f- actin interactions in cell motility. 1. To develop new microscopy methods for the simultaneous visualization and analysis of f-actin and microtubule dynamics and to carefully analyze microtubule/actin interactions in living migrating cells. 2. To biochemically and microscopically probe the molecular mechanism dynamic microtubule/actin interactions in Xenopus egg extracts. 3. To biochemically analyze microtubule mediation of Rho family signaling and study the mechanism by which growing microtubules activate Rac1 during cell motility. The results of these studies will greatly enhance our understanding of the mechanisms and mechanics of cell locomotion and will provide new methods for analyzing protein dynamics in living cells.